The largest database of trusted experimental protocols

82 protocols using H3K27ac

H3K27ac (39,133, Active Motif Lot # 31814008), H3K27ac (39,685, Active Motif, no. 14517014), H3K18ac (ab1191, Abcam, no. GR3211480–1), H3K27me3 (9756, Cell Signaling), H3K9ac (ab4441, Abcam), H4K16ac (39,167, Active Motif), H3 general (ab1791, Abcam, no. GR177884–2), Spike-In antibody (61,686, Active Motif, Lot# 00419007), p300 (sc-584, Santa Cruz, Lot # F3016), Gapdh (2118, Cell Signaling, Lot # 10), CBP(D6C5) (7389S, Cell Signaling), p53(CM5) (NCL-L-p53-CM5p, Leica Biosystems), PKCs p2056 (ab18192, Abcam), KAP1/TRIM29 pS824 (A300-767A, Bethyl), Acetyl-p53 (Lys379) (2570, Cell Signaling), anti-mouse IgG-HRP (NA93V, GE, no.9773218), anti-rabbit IgG-HRP (170–6515, Bio-Rad, no. 350003248).
+ Open protocol
+ Expand
Antibodies used were H3K27ac (1:50, Active Motif, 39133), H3K27ac (1:50, Active Motif, 91193), H3K27ac (1:50, AbCam, ab4729), H3K27me3 (1:50, Active Motif, 61017), Phospho-Rpb1 CTD (Ser2/Ser5) (1:50, Cell Signaling, 13546). For NTT-seq with surface markers readout on primary cells, the TotalSeq-A conjugated Human Universal Cocktail v1.0 panel was obtained from BioLegend (399907).
+ Open protocol
+ Expand
For immunoblotting, the following antibodies were used: NSD1 (NeuroMab: 75-280); NSD2 (Abcam:ab75359); NSD3 (Cell Signaling Technologies: 92056S); KLK3/PSA (Dako:A0562); FKBP5(Cell Signaling Technologies: 12210); NKX3-1 (Cell Signaling Technologies:83700S); FOXA1 N-terminal (Cell Signaling Technologies: 58613S; Sigma-Aldrich: SAB2100835); FOXA1 C-terminal (Thermo Fisher Scientific: PA5-27157); AR (Millipore: 06–680); AR (Abcam: ab133273); H3 (Cell Signaling Technologies: 3638S); GAPDH (Cell Signaling Technologies: 3683); H3K27me3(Millipore: 07–449); H3K36me2 (Cell Signaling Technologies: 2901S); H3K27Ac (Active Motif, Cat#39336); Phospho-AR (Ser-81) (Millipore, Cat# 07-1375-EMD); HALO (Thermo Scientific, Cat# G9281); HA (Cell Signaling Technologies, Cat# 3724S); His (Cell Signaling Technologies, Cat#2365). ChIP-seq assays were performed using the following antibodies: FOXA1 (Thermo Fisher Scientific: PA5-27157); AR (Abcam: ab133273); and H3K27Ac (Active Motif, Cat#39336).
+ Open protocol
+ Expand
Chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) was performed using 20 million cells per experiment. Cells were crosslinked for 7 min with 1% formaldehyde, then DNA was sonicated to an average fragment size of 200–500 base pairs using a Covaris M220 sonicator. Fragmented DNA was incubated with 10 µg of the following antibodies: Pol2 (Diagenode, C15200004), H3K27AC (Active Motif, 39133), H3K4me3 (Active Motif, 39915). Libraries were prepared using the NEBNext Ultra 2 kit (NEB, E7645S) and sequenced by the OGSR with >20 million reads per sample. Reads were filtered for quality by Trim Galore! (https://github.com/FelixKrueger/TrimGalore) and aligned to the hg38 genome with Bowtie2 [50 (link)], and visualized with SparK [51 ]. The location of putative G-quadruplex forming regions was determined using the G4P Calculator [52 (link)].
+ Open protocol
+ Expand
ChIP was performed as previously described (Letting et al., 2004 (link)). qPCR primers are listed in Table S2, antibodies are as follows: Brd4 (Bethyl A301–985A), H2AZ (Abcam Ab4174), total H3 (Abcam ab1791), H3S10ph (Abcam ab5176), H3K14ac (Millipore 07–353), H3K27ac (Active Motif 39685), H3K122ac (Abcam ab33309), H4K16ac (Millipore 07–370), and RNA Polymerase II (Cell Signaling 14958). ChIP-seq samples were prepared by end repair, 3 adenylation, and adaptor ligation using Illumina’s ChIP-seq Sample Preparation Kit. SPRIselect (Beckman Coulter) beads were used obtain an average library target size of 338 bp (range 302–436 bp), followed by real-time qPCR quantification using the KAPA Library Quant Kit for Illumina (KAPA Biosystems catalog no. KK4835). Single-end sequencing (1×50bp or 1×75 bp) was performed on the Illumina NextSeq 500 in high-output mode using Illumina sequencing reagents according to Illumina instructions, and bclfastq2 v 2.15.04 (default parameters) was used for conversion of reads to fastq. Fastq reads were aligned to the mm9 genome using bowtie 2 (parameters:–end-to-end–very-sensitive) (Langmead and Salzberg, 2012 (link)).
+ Open protocol
+ Expand
BT12 cells were incubated with 100 nM mithramycin or PBS control for 8 or 18 h. Cells were cross‐linked, lysed, and sheared as described (Harlow et al, 2019). 10 µg solubilized chromatin was immunoprecipitated with 1 µg mouse IgG and 1 µg H3K27me3 (Abcam); 2 µg rabbit IgG and 2 µg SMARCC1 (Cell Signaling); 1 µg rabbit IgG; and 1 µg H3K27ac (Active Motif). Antibody–chromatin complexes were immunoprecipitated and purified as described (Harlow et al, 2019). ChIP DNA was quantified with SYBR green relative to a standard curve generated with chromatin from the respective sample for each primer set. qPCR as described above was performed with the following primer sets (GAPDH, MYT1, SOX2, CCND1, SP1). Antibody information and PCR primer sequences are available in Appendix Table S5.
+ Open protocol
+ Expand
ChIP was performed as previously described (Letting et al., 2004 (link)). qPCR primers are listed in Table S2, antibodies are as follows: Brd4 (Bethyl A301–985A), H2AZ (Abcam Ab4174), total H3 (Abcam ab1791), H3S10ph (Abcam ab5176), H3K14ac (Millipore 07–353), H3K27ac (Active Motif 39685), H3K122ac (Abcam ab33309), H4K16ac (Millipore 07–370), and RNA Polymerase II (Cell Signaling 14958). ChIP-seq samples were prepared by end repair, 3 adenylation, and adaptor ligation using Illumina’s ChIP-seq Sample Preparation Kit. SPRIselect (Beckman Coulter) beads were used obtain an average library target size of 338 bp (range 302–436 bp), followed by real-time qPCR quantification using the KAPA Library Quant Kit for Illumina (KAPA Biosystems catalog no. KK4835). Single-end sequencing (1×50bp or 1×75 bp) was performed on the Illumina NextSeq 500 in high-output mode using Illumina sequencing reagents according to Illumina instructions, and bclfastq2 v 2.15.04 (default parameters) was used for conversion of reads to fastq. Fastq reads were aligned to the mm9 genome using bowtie 2 (parameters:–end-to-end–very-sensitive) (Langmead and Salzberg, 2012 (link)).
+ Open protocol
+ Expand
ChIP assays were performed as previously described17 (link),18 (link),63 (link),64 (link) for the following histone marks: H3K4me3 (Abcam, #ab8580), H3K4me1 (Active Motif, #39297), H3K36me3 (Abcam, #ab9050), H3K27ac (Active Motif, #39133), H2AZac (Abcam, #ab18262), H3K9ac (Millipore, #06-599) and H3K27me3 (Millipore, #07-449). ChIP of H3K9me3 (Diagenode, #C15500003) was performed as previously described65 (link). We performed lamin ChIP assays in PrEC and LNCaP as previously described66 (link) for both Lamin B1 (Abcam, #ab16048) and Lamin A/C (Santa Cruz, #sc7292). Each ChIP assay was validated by qPCR against an IgG control and enrichment above input. Libraries were prepared with the Illumina TruSeq Chip Library Prep Kit and sequenced on an Illumina HiSeq 2500.
Sequencing data was processed as previously described17 (link),63 (link). Briefly, ChIP-seq reads were aligned to hg19 using bowtie58 (link) (v1.1.0) allowing up to 3 mismatches, discarding ambiguous and clonal reads. All histone ChIP-seq peaks were called using PeakRanger67 (v1.16). Broad domains of lamins (LADs), H3K9me3 and H3K27me3 were called using the enriched domain detector (EDD) for identification of wide genomic enrichment domains68 (link).
+ Open protocol
+ Expand
Western blotting was performed using standard procedures. Primary antibodies are as follows: Brd4 (Bethyl A301–985A), H2AZ (Active Motif 39113), total H3 (Abcam ab1791), H3S10ph (Abcam ab5176), H3K14ac (Millipore 07–353), H3K18ac (Millipore 07–354), H3K27ac (Active Motif 39134), H3K122ac (Abcam ab33309), H4K16ac (Millipore 07–370), GAPDH (Santa Cruz sc-365062). Secondary antibodies included LI-COR IRDye 800 CW (Donkey anti-mouse, Donkey anti-rabbit) and LI-COR IRDye680 (Donkey anti-mouse, Donkey anti-rabbit). Imaging performed on the LI-COR Odyssey. Quantification performed within ImageStudio by subtracting background signal for each band and subsequently normalizing to GAPDH intensity.
+ Open protocol
+ Expand
Reduced samples were loaded onto NuPAGE Bis-Tris (Thermo Fisher Scientific) gels in MOPS buffer and separated proteins were transferred onto a PVDF membrane for immunodetection with anti-PPARγ (C26H12, 1:2000: Cell Signaling Technologies, Danvers, MA, USA), HSP90α (GTX109753: Gene Tex, Irvine, CA, USA, 1:2000), HDAC1 (GTX100513: Gene Tex, 1:2000), GAPDH (GTX100118: Gene Tex, 1:1000), H3K27ac (39134: Active Motif, Carlsbad, CA, USA, 1:2000), H3K9ac (39918: Active Motif, 1:2000), H3k27me3 (C36B11: Cell Signaling Technologies, 1:2000), and Histone H3 (D2B12: Cell Signaling Technologies, 1:2000) antibodies as primary antibodies, and the HRP-conjugated goat anti-rabbit antibody (#7074: Cell Signaling Technologies, 1:2000) as the secondary antibody.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!